One year of experience with combined pharmacokinetic/pharmacogenetic monitoring of anti-TNF alpha agents: a retrospective study

Anti-tumor necrosis factor alpha (anti-TNFα) inhibitors are used extensively for the management of moderate to severe inflammatory bowel disease (IBD) in both adult and pediatric patients. Unfortunately, not all patients show an optimal response to induction therapy, while others lose their response over time for reasons yet poorly understood. We report on a pharmacokinetic/pharmacogenetic approach to monitor the therapy with anti-TNFα in a real-world cohort of seventy-nine pediatric patients affected by IBD that was analyzed retrospectively. We evaluated plasma concentrations of infliximab, adalimumab, and related anti-drug antibodies (ADAs), and single nucleotide polymorphisms (SNPs) in genes involved in immune processes and inflammation on the anti-TNFα response. We found a significant association between the SNP in TNFα promoter (−308G>A) and clinical remission without steroids in patients on infliximab therapy. Additionally, a potential connection between HLA-DQA1*05 genetic variant carriers and a higher risk of anti-TNFα immunogenicity emerged.


INTRODUCTION
In the last decades, the prevalence of inflammatory bowel diseases (IBD) in children has increased significantly, with an incidence of ~8-10 cases per 100,000 people per year in Western countries [1,2].Crohn's disease (CD) and ulcerative colitis (UC), the two main clinical manifestations of IBD, are characterized by a chronic and extremely variable relapsing course composed of alternating phases of remissions and exacerbations [3].Pharmacological therapy of IBD, and in particular the use of antibodies against tumor necrosis factor alpha (anti-TNFα), such as infliximab and adalimumab, has greatly improved the clinical outcomes [4,5].The therapy with anti-TNFα agents has been shown not only to induce but also to maintain steroid-free remission, and reduce the need for surgery [6,7], although in the presence of a relevant inter-individual variability in the response, with 20-40% of IBD patients losing the response to anti-TNFα induction therapy and ~50% of primary responders subsequently losing it during the maintenance phase [8,9].In addition, some patients develop antidrug antibodies (ADAs) with potential risk of immunogenicity and loss of response.
Therapeutic drug monitoring (TDM) has emerged as a useful tool for optimizing the use of anti-TNFα therapies in IBD, especially when used in a proactive (instead of reactive) way [9][10][11][12].Better therapeutic outcomes have been observed when proactive utilization of TDM for infliximab and adalimumab was preferred over conventional management in patients with IBD [13][14][15][16].Proactive TDM can also efficiently guide infliximab therapy by minimizing under-treatment, reducing over-treatment, allowing early identification of ADAs development, enabling the detection of therapeutic failures, stopping ineffective treatment timely and the prevention of hypersensitivity reactions [13][14][15][16].
Genetic variability may have an important role in the clinical evolution of IBD patients; some authors have recently reported the effects of single nucleotide polymorphisms (SNPs) in genes involved in immune processes and inflammation on the efficacy of anti-TNFα agents, including in IBD [17][18][19][20][21].Many variants in the untranslated region of the human TNFα gene have been described in adult patients affected by IBD that have variously been reported to alter the transcriptional process, thus influencing TNFα production [22,23].SNPs identified in the promoter and receptor regions of the TNFα gene have been shown to influence anti-TNFα treatment response in IBD patients [24][25][26].Increasing evidence suggests the existence of association between genetic variation in the class II human leucocyte antigen (HLA) gene region (HLA-DQA1*05) and an increase in the risk of immunogenicity to both infliximab and adalimumab in patients with IBD [27,28].Currently, there are no diagnostic tools able to predict which patients will develop ADAs, become unresponsive to therapy, or develop an anti-TNFα hypersensitivity response.
In this study, we aimed to identify predictive pharmacokinetic and/or pharmacogenetic biomarkers able to capture substantial changes in the clinical outcomes of the enrolled patients.

MATERIALS AND METHODS Patients methods and study design
We performed a retrospective analysis of routine requests for TDM analysis of infliximab and adalimumab (also including samples sent by other hospitals) received as a diagnostic service by the Unit of Clinical Pharmacology at the Luigi Sacco University Hospital (Milan, Italy), between July 2020 and July 2021.Patients aged 6-18 years previously diagnosed with CD or UC according to the European Society Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) revised Porto criteria [29], and treated with infliximab or adalimumab were considered in this retrospective analysis.In these patients, an anti-TNFα therapy has been started according to ESPGHAN guidelines [30,31].Demographic and clinical data, including age at diagnosis, localization and behavior of disease according to the Paris classification [32], and any previous therapies, were collected.For each patient, clinical activity was assessed, using the Pediatric Crohn's Disease Activity Index (PCDAI) and Pediatric Ulcerative Colitis Activity Index (PUCAI) for patients with CD and UC respectively, at the start of anti-TNFα therapy and the end of follow up.Disease was considered in clinical remission if indexes were less than or equal to 10. PCDAI > 40 and PUCAI > 65 at diagnosis were used to define severe disease.The responsiveness was defined as: 1-sustained clinical remission (PCDAI ≤ 10 points) without steroids over all visits starting from week 14 for infliximab and week 8 for adalimumab to the last follow-up (up to 5 years maximum) for both; 2-sustained biologic remission (defined by CRP ≤ 0.5 mg/dL and fecal calprotectin ≤150 μg/g) from week 14 (for infliximab) and from week 8 (for adalimumab) to the last follow up (up to 5 years maximum).
We retrieved also data on serum hemoglobin, C reactive protein (CRP), erythrocyte sedimentation rate (ESR), albumin, and fecal calprotectin carried out in all patients.Information on the clinical management of the patients (i.e., treatment intensification, agents like steroids, non-steroid anti-inflammatory drugs, i.e., mesalazine, azathioprine) were also collected.
Infliximab was given intravenously in standard dosing (5 mg/kg) weightbased during the induction (infusion at weeks 0, 2, and 6).Subsequently, the dosing regimen was decided by the clinician on target trough concentrations or clinical status (during maintenance phase, every 4/ 8 weeks) [33].Adalimumab was administered via subcutaneous injection on day 1 at 160 and 80 mg, for body weight ≥40 or ≤40 kg, respectively (induction phase); followed by every other week at 40 or 20 mg for body weight ≥40 or ≤40 kg, respectively (maintenance phase).Adalimumab treatment may be escalated when there is clinical indication of an inadequate response by reducing the time between injections to once a week.
Pharmacogenetic analyses were conducted retrospectively to assess the effect of the polymorphisms in the TNFα and HLA-DQA1 regions on efficacy clinical outcomes.Patients were stratified into two groups according to their treatment: group 1 treated with infliximab (IFX) and group 2 with adalimumab (ADM).The two groups were analyzed together (IFX + ADM) to assess the association of HLA-DQA1 variants (HLA-DQA1*05 allele and SNP rs2097432) with immunogenicity risk in IBD cohort.

Assessment of plasma drug concentrations
Plasma samples for anti-TNFα measurements, infliximab, and adalimumab, were performed by a lateral flow immunochromatographic assay with the Ridaquick Monitoring assay (R-Biopharm AG, Darmstadt, Germany).The measurement range was from 0.5 to 10 mg/L with the use of the standard dilution (maintenance therapy phase) and can be extended to 2-40 mg/L through an additional dilution.The concentration of the two anti-TNFα was measured at trough, before the next scheduled administration.A target therapeutic trough concentration window of 3-7 mg/L is recommended for infliximab [33], and 5-10 mg/L for adalimumab in IBD [11].ADAs were measured when anti-TNFα plasma levels were less than or equal to 1.0 mg/ L with an enzyme-linked immunosorbent assay (ELISA) based method with the Ridascreen kit (R-Biopharm AG, Darmstadt, Germany).The assay was performed according to the manufacturer's guidelines.Samples were measured in a 1/25 dilution (LLOQ 2.5 ng/mL; HLOQ 125 ng/mL) or a 1/200 dilution (LLOQ 20 ng/mL; HLOQ 1000 ng/mL).Only for infliximab, a cutoff of 119 ng/mL was determined to discriminate between high and low ADA concentrations, a parameter that may help clinicians make treatment decision [34].

Genotyping
Genomic DNA was isolated from peripheral blood cells using an automated DNA extraction system (EZ1 Advanced XL, Qiagen, Hilden, Germany) according to the manufacturer's instructions.DNA concentration and purity were evaluated by absorbance methodology using a NanoDrop 1000 Spectrophotometer V3.7 (Thermo Fisher Scientific, Waltham, Massachusetts).All subjects were genotyped for three TNFα gene polymorphisms (−238G>A, −308G>A, and −857C>T) and one SNP (rs2097432) in the HLA-DQA1 region.All genotypes were determined by Real-Time PCR using the LightSNiP (TIB-MolBiol, Berlin, Germany) on a LightCycler 480 (Roche, Basel, Switzerland), according to the manufacturer's instructions.Data analysis was performed as described in the LightCycler 480 Instrument operator's manual.Genotyping performance was estimated through the use, in each analysis, of known-genotype internal quality controls.
The HLA-DQA1*05 allele was identified using a TaqMan allelic discrimination assay with a Genvinset® HLA Celiac Plus diagnostic kit (provided by Nuclear Laser Medicine srl, Milan, Italy), according to the manufacturer's instructions.

Ethics statement
Data used for this study were collected for clinical purposes and were previously anonymized, according to the requirements set by the Italian Data Protection Code (leg.Decree 196/2003) and by the general authorizations issued by the Data Protection Authority.All patients were registered in the IBD Registry of the Italian Society of Pediatric Gastroenterology, Hepatology and Nutrition (SIGENP).The methodology of the registry has been previously described in detail [35].The study was conducted in accordance with the Declaration of Helsinki, and all patients and parents and/or legal guardians of subjects who are under 18 provided written informed consent.

Statistical methods
Quantitative values were given as mean ± standard deviation or as the median and interquartile range (IQR) based on the distribution of the data verified through the Kolmogorov Smirnov test, whereas qualitative variables were described as counts and relative frequency (%) of each category.Comparisons between groups were made with a chi-square test for qualitative variables and with a t-test or Mann-Whitney test for quantitative data.Significance was set at p < 0.05.Associations between quantitative variables were assessed with the Pearson correlation coefficient (Pearson's r).All genotypes were tested for Hardy-Weinberg equilibrium using χ 2 test.Statistical comparisons for genotype frequencies between the two groups were tested by χ 2 test.Statistical significance was defined at two-sided p values <0.05.We used the odds ratio (OR) with 95% confidence intervals (95% CI) as a measure of the effect of risk genotype on each endpoint.Associations between clinical response and demographic and clinic-pathological characteristics, chemotherapy regimens, and genotypes were assessed using the χ 2 test, Fisher's exact test or Wilcoxon signed rank test, as appropriate.

Patient population
Of the 82 patients, 3 were excluded from the analysis due to incomplete clinical information.Seventy-nine patients (57 with CD and 22 with UC) treated with anti-TNFα agents (infliximab and adalimumab) were included in the study.The demographic and clinical characteristics of the patient population are summarized in Table 1.Fifty-seven patients were on infliximab therapy (145 measurements), and twenty-two were on adalimumab (31 dosages).Proactive TDM was utilized by approximately 80% of physicians.TDM was performed twice (81% for IFX and 39% for ADM), thrice (53% for IFX and 17% for ADM), four (26 and 4%), five (9% for IFX only), six (5% for IFX only) times a year.Infliximab was stopped because of: adverse events (dermatological) in 1 (1.7%) patient; loss of response due to anti-infliximab antibodies in 5 (8.7%) patients; and loss of response through ineffectiveness in 1 (1.7%) patient.Adalimumab was stopped in 2 (9%) patients for loss of response during the maintenance phase and for the ADAs development in 3 (13.6%)patients.Dose optimization was applied in 24 out of the 57 patients (42.1%) in the infliximab group (increased dose, n = 16; increased frequency of drug administration, n = 7; both actions, n = 1), and in 4 out of 22 patients (18.18%) in the adalimumab group (increased frequency of drug administration, n = 4).At the inception of the therapy with infliximab, 11 (19.3%)patients were in clinical remission according to the PCDAI/PUCAI activity score, yet had persistent biochemical alterations (elevated CRP and fecal calprotectin levels) (Table 2).After optimization, 50 patients (87.7%) were in clinical remission, but only 26 (45.6%) without an abnormal biomarker (CRP ≤ 0.5 mg/dL and fecal calprotectin ≤150 μg/g).Finally, at the last follow-up, out of 50 patients in clinical remission, 34 (59.6%) had normal biomarker values.No statistical association between biochemical variables and clinical response was found at any time point.At the start of the therapy with adalimumab, about half (45.5%) of the patients were in clinical remission but only three (13.6%)had not an abnormal biomarker (Table 2).At the last follow-up, 16 (73%) patients were in clinical remission but no more than seven (32%) had normal biomarkers.Infliximab and adalimumab plasma levels from baseline to the last follow-up are shown in Fig. 1; as can be seen, 87% of patients on either drug were on therapeutic target at the beginning of therapy.In summary, the median concentrations of both drugs were higher during induction (12.6 ± 12 mg/L for IFX, and 11.5 ± 8 mg/L for ADM), as required by the recommendations [36], than during maintenance (>3 mg/L for IFX and >5 for adalimumab).There was no association between the concentrations and disease activity.

Association between TNFα candidate variants and clinical response
The genotype distribution of the SNPs in the TNFα and HLA-DQA1 genes is summarized in Supplementary Table 1.Genotype frequencies are in Hardy-Weinberg equilibrium (HWE; p > 0.05).
Clinical remission without steroids occurred in 50.6% of the patients in the infliximab group and in 59% in the adalimumab one.A significant association between the TNFα rs1800629 (−308G>A) and sustained without steroids clinical remission was found in the infliximab group at the last follow-up (p = 0.03, odds ratio [OR] GG vs. GA+AA genotype = 12.6, [CI] = 1.28-124.5).No   3).
In the adalimumab group, there was no association between any SNPs in the TNFα gene and the clinical response (Table 3).Furthermore, no association with sustained biological remission was found for the infliximab or adalimumab groups or TNFα SNPs.The effects of all covariates (population demography and clinical characteristics) could not be verified due to the limited sample size of these subgroups.

Association between allelic variation in HLA-DQA1 gene and immunogenicity
Seventy-nine patients were screened for the HLA-DQA1*05 variant allele and the HLA-DQA1 rs2097432T>C polymorphism.The HLA-DQA1*05 allele was found to be common in our population, with a frequency of 68%; the genetic variation (rs2097432) was also relatively common, with a minor allele frequency of 56%.HLA-DQA1 rs2097432T>C was in Hardy-Weinberg equilibrium (Supplementary Table 1).HLA-DQA1 variant carriers (TC + CC genotype) had a significant association with ADAs formation (p = 0.04 chisquare test, not shown) but the likelihood that a patient with the HLA-DQA1 single-nucleotide variation will develop antibodies is not significant (Table 4: OR = 6.7, 95% CI = 0.79-56, p = 0.07).Also, carriers of the HLA-DQA1*05 allele have no significant association with the risk of developing antibodies anti-TNFα (Table 4: OR = 4.1, 95% CI = 0.49-35, p = 0.19).
Moreover, eight (10.1%)out of the seventy-nine individuals developed antibodies, three of these had CC genotype with very high antibody titer (>500 ng/mL), five patients had TC genotype (three with low antibody titers of <119 and two with high titer); and finally, one patient with the wild type genotype who developed antibodies had a low antibody titer.

DISCUSSION
In the current study, we report on a retrospective analysis of the value of an approach combining pharmacokinetic/pharmacogenetic monitoring of the anti-TNFα agents obtained over a one-year period in pediatric patients.In our pediatric cohort, anti-TNFα therapy showed a high initial response rate, but up to 20% of patients in the maintenance phase required a dose adjustment to sustain a clinical response.The clinical scenarios revealed that physicians generally continued the therapeutic regimen in the presence of therapeutic drug levels, increased the dose in the presence of sub-therapeutic drug concentrations, and switched the drug within class in the presence of neutralizing ADAs, with a minority preferring dose intensification or optimization of concomitant immunosuppression in an effort to overcome ADAs.Although we did not find significant associations between TNFα trough concentrations and disease activity, possibly due to the use of proactive TDM, it is important to note that this is a retrospective analysis of data collected in real-life settings, where there wasn't a control group to evaluate the effect of the dose optimization.We can only assume the application of proactive TDM may have contributed to optimizing clinical remission rates and biomarker levels.Several studies in the literature report an anti-TNFα treatment failure associated with suboptimal drug concentrations, which suggests the possibility of improving IBD patients outcomes by optimizing their therapy [9][10][11][12][13].Our study suggests a potential role for the TNFα SNP rs1800629 (−308G>A) as a useful genetic biomarker of the response to infliximab in IBD.In particular, the pharmacogenetic analyses showed that patients with the TNFα  −308G common allele had increased rates of sustained clinical remission without steroids infliximab therapy compared with patients with the variant allele (TNFα −308A).Cumulative corticosteroid exposure from prolonged use or repeated courses might cause growth stunting, hyperglycemia, cushingoid characteristics, skin and mood changes, increased infection risk, and decreased bone density in children with IBD [37].In an effort to reduce the utilization and adverse reactions from corticosteroid treatment, current professional guidelines recommend steroidsparing regimens, which can be effective for many people with IBD [38,39].The association between the TNFα SNP rs1800629 (−308G>A) and response to anti-TNFα agents in inflammatory disorders has been the subject of several investigations with conflicting results [26,[40][41][42][43].In rheumatoid arthritis, spondyloarthropathy, and IBD meta-analyses, the TNFα −308A allele carriers were found to respond less to anti-TNFα drugs [41][42][43].Genetic changes introduced by this variant could potentially affect the binding of transcription factor, thus altering the activity of the promoter and the resulting mRNA and protein levels [44,45], at last increasing TNFα production.These data are, however, from adult patients whereas those available for pediatric patients are even scarcer.Recently, Curci et al. evaluated the impact of SNP rs396991 in FCGR3A (Fc Gamma Receptor IIIa) and rs1800629 in TNFα genes on infliximab response in an Italian cohort of 76 pediatric patients [26].In this study, they only found an association between the FCGR3A SNP, infliximab response, and ADAs production.We have previously described a case of increased anti-TNFα drug clearance associated with rapid loss of response without the development of specific ADAs that cannot be explained by clinical covariates alone [46].In that case report, pharmacogenetic analysis revealed that patient was homozygous for the TNFα −308A rare variant, suggesting an influence of this polymorphism in the observed fast clearance of infliximab and adalimumab due to increased TNFα production.Another important cause of loss of response in patients with IBD is the production of antibodies against anti-TNFα drugs [47].The formation of ADAs is a major contributory factor to inactivation by increasing clearance of anti-TNFα agents.We did not find an association between the variant allele V (HLA-DQA1*05) and a higher probability of ADAs formation, as instead previously demonstrated [26].This is possibly due to the high frequency of the allele found in our population and the concomitant use of immunomodulators (which reduce the risk of immunogenicity).Consistent with previous studies in adult patients with IBD treated with infliximab and adalimumab [27,28], we found an association between the HLA-DQA1 rs2097432 SNP and ADAs formation; however, the high frequency of this SNP is not a good indicator of the immunogenicity risk in our cohort.The current study is limited by several factors, particularly the small sample size, which limits its statistical power.Furthermore, due to the real-life setting of this study, without a comparison group, it was impossible to evaluate the different variables (serum concentrations, biomarker levels, etc.) over the course of the treatment.Further study in a larger cohort of pediatric patients with IBD may help better delineate the contribution of TNFα SNP in response to anti-TNFα and the HLA-DQA1 genetic variant on ADAs production.
In conclusion, despite the small sample size and its retrospective design, the present study has identified some useful markers to improve drug tolerability and efficacy in pediatric IBD patients.The SNPs in TNFα and the HLA-DQA1 gene can be helpful to tailor the anti-TNFα treatment, especially for individuals who need more frequent clinical and biochemical monitoring and a proactive TDM approach to enhance the clinical response.The combined pharmacokinetic/pharmacogenetic monitoring of anti-TNF alpha agents is a further step towards the personalization of the therapy, which should integrate different approaches and evaluations to obtain the maximum benefit for the patient.

Fig. 1
Fig. 1 Infliximab and adalimumab concentrations over time.Infliximab (A) and adalimumab (B) plasma concentrations by time, from baseline to the last follow-up (for A, n = 145 measurements; for B, n = 31 measurements).Box plot values include: the lower adjacent value, the first quartile, the median, the third quartile, and the upper adjacent value (in ascending order).
N number, SD standard deviation, IQR interquartile range.a L4 is a modifier that can be added to L1-L3 when concomitant upper gastrointestinal disease is present.P # is added to B1-B3 when concomitant perianal disease is present.

Table 2 .
Characteristics of patients by time.ESR erythrocyte sedimentation rate, CRP C reactive protein, N number, SD standard deviation, ND not available.

Table 3 .
Associations between the TNFα polymorphisms and clinical response.CRP C reactive protein.Bold text indicates a statistically significant difference with a p-value less than 0.05.

Table 4 .
Association between HLA-DQA1 allele, SNP, and development of anti-drug antibodies to anti-TNFα.